Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment.

BACKGROUND: Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs. METHODS: Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program. RESULTS: We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133(pos)CD45(neg) (5 ± 1) and (2 ± 1) and CK20(pos)CD45(neg) of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4(pos) of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded-CTCs (eCTCs). ECTCs organized in tumor sphere were CD45(neg)CD133(pos) while in adhesion were CXCR4(pos)CK20(pos). These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45(neg)CD133(pos)) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4(pos)CK20(pos)) have a low disease-free survival. CONCLUSION: On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic-risk personalized score thus improving disease management and reducing patient care cost.

Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma.

The Hedgehog (Hh) pathway is required for cell-fate determination during the embryonic life, as well as cell growth and differentiation in the adult organism, where the inappropriate activation has been implicated in several cancers. Here we demonstrate that Hh signaling plays a significant role in growth and survival of multiple myeloma (MM) cells. We observed that CD138(+) MM cells express Hh genes and confirmed Smoothened (Smo)-dependent Hh signaling in MM using a novel synthetic Smo inhibitor, NVP-LDE225 (Novartis), which decreased MM cell viability by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of Hh activity. In addition, we detected a nuclear localization of Gli1 in MM cells, which is completely abrogated by Forskolin, a Gli1-modulating compound, confirming Smo-independent mechanisms leading to Hh activation in MM. Finally, we identified that bone marrow stromal cells are a source of the Shh ligand, although they are resistant to the Hh inhibitor because of defective Smo expression and Ptch1 up-regulation. Further in vitro as well as in vivo studies showed antitumor efficacy of NVP-LDE225 in combination with bortezomib. Altogether, our data demonstrate activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for testing Hh inhibitors in clinical trials to improve MM patient outcome.

In vivo activity of miR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p<0.05) which translated in mice survival benefits (p=0.0047). Analysis of miR-34a and NOTCH1 expression in tumor retrieved from animal demonstrated efficient delivery and gene modulation induced by SNALPs miR-34a in the absence of systemic toxicity. We here therefore provide evidence that SNALPs miR-34a may represent a promising tool for miRNA-therapeutics in MM.

Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth.

PURPOSE: Deregulated expression of miRNAs plays a role in the pathogenesis and progression of multiple myeloma. Among upregulated miRNAs, miR-21 has oncogenic potential and therefore represents an attractive target for the treatment of multiple myeloma. EXPERIMENTAL DESIGN: Here, we investigated the in vitro and in vivo anti-multiple myeloma activity of miR-21 inhibitors. RESULTS: Either transient-enforced expression or lentivirus-based constitutive expression of miR-21 inhibitors triggered significant growth inhibition of primary patient multiple myeloma cells or interleukin-6-dependent/independent multiple myeloma cell lines and overcame the protective activity of human bone marrow stromal cells. Conversely, transfection of miR-21 mimics significantly increased proliferation of multiple myeloma cells, showing its tumor-promoting potential in multiple myeloma. Importantly, upregulation of miR-21 canonical validated targets (PTEN, Rho-B, and BTG2), together with functional impairment of both AKT and extracellular signal-regulated kinase signaling, were achieved by transfection of miR-21 inhibitors into multiple myeloma cells. In vivo delivery of miR-21 inhibitors in severe combined immunodeficient mice bearing human multiple myeloma xenografts expressing miR-21 induced significant antitumor activity. Upregulation of PTEN and downregulation of p-AKT were observed in retrieved xenografts following treatment with miR-21 inhibitors. CONCLUSION: Our findings show the first evidence that in vivo antagonism of miR-21 exerts anti-multiple myeloma activity, providing the rationale for clinical development of miR-21 inhibitors in this still incurable disease.

In vitro and in vivo anti-tumor activity of miR-221/222 inhibitors in multiple myeloma.

A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/ NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.

Synthetic miR-34a mimics as a novel therapeutic agent for multiple myeloma: in vitro and in vivo evidence.

PURPOSE: Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. EXPERIMENTAL DESIGN: Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. RESULTS: Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. CONCLUSIONS: Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a-based treatment strategies in MM patients.